U.S. patent application number 12/223276 was filed with the patent office on 2011-02-17 for inhibiting amyloid-beta peptide/rage interaction at the blood-brain barrier.
Invention is credited to Rashid Deane, Benjamin L. Miller, Berislav V. Zlokovic.
Application Number | 20110039908 12/223276 |
Document ID | / |
Family ID | 38327922 |
Filed Date | 2011-02-17 |
United States Patent
Application |
20110039908 |
Kind Code |
A1 |
Zlokovic; Berislav V. ; et
al. |
February 17, 2011 |
Inhibiting Amyloid-Beta Peptide/Rage Interaction At The Blood-Brain
Barrier
Abstract
Small molecules are used to inhibit specific receptor-ligand
interaction between Alzheimer's amyloid-.beta. peptide (A.beta.)
and Receptor for Advanced Gly-cation Endproducts (RAGE). Objectives
include treating Alzheimer's disease and other pathologies
involving cerebral amyloid angiopathy; improving blood flow to or
within the brain; decreasing the level of A.beta. in the brain;
reducing neuropathology associated with Alzheimer's disease;
reducing inflammation and/or oxidant stress in the brain; improving
memory and/or learning; treating other conditions involving
A.beta./RAGE interaction at the blood-brain barrier, RAGE-mediated
transport of A.beta. into the brain, or RAGE activation in brain
vasculature and/or brain parenchyma (e.g., diabetic complications);
or any combination thereof.
Inventors: |
Zlokovic; Berislav V.;
(Rochester, NY) ; Deane; Rashid; (Rochester,
NY) ; Miller; Benjamin L.; (Penfield, NY) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Family ID: |
38327922 |
Appl. No.: |
12/223276 |
Filed: |
January 26, 2007 |
PCT Filed: |
January 26, 2007 |
PCT NO: |
PCT/US2007/002220 |
371 Date: |
July 25, 2008 |
Current U.S.
Class: |
514/415 ;
514/427; 514/616; 514/617; 548/504; 548/561; 564/155; 564/184 |
Current CPC
Class: |
A61K 31/167 20130101;
A61K 31/166 20130101; A61P 43/00 20180101; A61P 25/28 20180101 |
Class at
Publication: |
514/415 ;
548/504; 514/427; 548/561; 514/616; 564/155; 514/617; 564/184 |
International
Class: |
A61K 31/404 20060101
A61K031/404; C07D 209/14 20060101 C07D209/14; A61K 31/40 20060101
A61K031/40; C07D 207/335 20060101 C07D207/335; A61K 31/167 20060101
A61K031/167; C07C 235/84 20060101 C07C235/84; A61K 31/166 20060101
A61K031/166; C07C 233/64 20060101 C07C233/64; A61P 25/28 20060101
A61P025/28 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 26, 2006 |
US |
60762117 |
Claims
1. A method of inhibiting specific receptor-ligand interaction
between Alzheimer's amyloid-.beta. peptide (A.beta.) and Receptor
for Advanced Glycation Endproducts (RAGE) or RAGE activation,
comprising administering an effective amount of one or more
tertiary amides of formula R.sub.1(CO)NR.sub.2R.sub.3 such that at
least some A.beta./RAGE interaction is inhibited; wherein R.sub.1
is an electron-deficient aryl moiety, R.sub.2 is a hydrophobic
hydrocarbon moiety, and R.sub.3 is an electron-rich aryl moiety
connected directly or by an optional linker.
2. The method according to claim 1, wherein R.sub.1 is an aryl
moiety substituted with one or more electron withdrawing
groups.
3. The method according to claim 1, wherein R.sub.2 is a straight
or branched aliphatic, alicyclic, straight or branched acyclic or
cyclic olefin, straight or branched acyclic or cyclic acetylene,
aryl-containing, and/or amine-containing C4 to C18 hydrocarbon
moiety which is optionally substituted.
4. The method according to claim 1, wherein R.sub.3 is a furanyl,
pyrrolyl, benzyl, phenyl, alkylphenyl, alkoxy-phenyl, naphthyl,
benzofuryl, indyl, or quinolyl moiety which is optionally
substituted.
5. The method according to claim 4, wherein R.sub.3 is linked by a
C1 to C4 alkyl or heteroaromatic linker.
6. The method according to claim 1, wherein the one or more
tertiary amides are selected from the group consisting of compounds
illustrated in FIGS. 1 and 9.
7. The method according to claim 1, wherein the one or more
tertiary amides are administered in vivo to a subject in need of
treatment as a pharmaceutical composition.
8. The method according to claim 7, wherein at least some
RAGE-mediated A.beta. transport into the subject's brain is
inhibited by treatment.
9. The method according to claim 7, wherein at least some RAGE
activation by A.beta. at the subject's blood-brain barrier is
inhibited by treatment.
10. The method according to claim 7, wherein at least A.beta. is
decreased in its amount or concentration in the subject's brain by
treatment.
11. The method according to claim 10, wherein at least
neuropathology associated with Alzheimer's disease is reduced in
the subject's brain by treatment.
12. The method according to claim 7, wherein at least cerebral
blood flow is increased in the subject by treatment.
13. The method according to claim 7, wherein memory and/or learning
is improved in the subject by treatment.
14. The method according to claim 7, wherein at least some
A.beta.-RAGE interaction at the subject's blood-brain barrier is
disrupted.
15. A compound selected from the group consisting of compounds
illustrated in FIGS. 1 and 9.
16. A composition comprising (i) one or more of the compounds of
claim 15 and (ii) at least one vehicle or carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of provisional Appln.
No. 60/762,117, filed Jan. 26, 2006.
BACKGROUND OF THE INVENTION
[0002] The invention relates to inhibiting specific receptor-ligand
interaction between Alzheimer's amyloid-.beta. peptide (A.beta.)
and the Receptor for Advanced Glycation Endproducts (RAGE).
[0003] A number of genetic, cellular, biochemical, and animal
studies suggest accumulation of amyloid .beta.-peptide (A.beta.) in
the brain is the key event in Alzheimer's disease (AD), while the
rest of the disease process, including formation of neurofibrillar
tangles results from an imbalance between A.beta. production and
A.beta. clearance (Hardy & Selkoe, 2002; Tanzi et al., 2004;
Zlokovic, 2005). A.beta. is neurotoxic (Walsh et al., 2002; Kayed
et al., 2003; Gong et al., 2003) and deposits as amyloid in brain
parenchyma and brain vessels in patients with sporadic AD and
familial forms of AD (FAD). The mechanisms responsible for A.beta.
production, i.e., the proteolytic enzymes .beta.- and
.gamma.-secretases which cleave A.beta. from its larger precursor
protein (APP), have been characterized (Selkoe, 1998; Vassar et
al., 1999) and their respective inhibitors developed. Increased
A.beta. production, however, can explain only a small number of
early onset FAD cases bearing inherited mutations in the APP gene
(i.e., Swedish mutation) or presenilins 1 or 2 genes, but does not
contribute to late-onset AD or >98% of all AD cases (Selkoe,
2001; Holtzman & Zlokovic, 2006). According to a new emerging
concept, reduced A.beta. clearance and/or its increased influx and
re-entry into the brain from circulation via transport across the
blood-brain barrier (BBB) may be responsible for A.beta. brain
accumulations in sporadic AD (Tanzi et al., 2004, Zlokovic, 2005;
Holtzman & Zlokovic, 2006).
[0004] Recent evidence indicates that A.beta. within the
intravascular space is linked to deposited A.beta. in the brain
suggesting that transport of A.beta. from blood to brain and from
brain to blood across the blood-brain barrier (BBB) regulates brain
A.beta. (Shibata et al., 2000; De Mattos et al., 2002a; De Mattos
et al., 2002b; Bading et al., 2002; Mackic et al., 2002; Carro et
al., 2002; Deane et al., 2003; Deane et al., 2004; Tanzi et al.,
2004; Zlokovic, 2005; Holtzman & Zlokovic, 2006). Numerous
studies in animal models (reviewed by Deane et al., 2004; Holtzman
& Zlokovic, 2006) and some studies in AD patients demonstrating
increased levels of A.beta. on plasma lipoproteins and proteins
(Matsubara et al., 1999; Kuo et al., 1999), have suggested that
re-entry of circulating A.beta. into the brain via transport across
the BBB is an important source of brain A.beta.. High plasma levels
of A.beta.40/42 have been determined in mouse models of A.beta.
under basal conditions, e.g., APPsw.sup.+/- mice (Kawarabayashi et
al., 2001) or after treatment with A.beta. peripheral binding
agents, e.g., anti-A.beta. antibodies (De Mattos et al., 2002a),
sRAGE (Deane et al., 2003), which confirms the link between
intravascular and brain A.beta..
[0005] RAGE is a multiligand receptor in the immunoglobulin
superfamily which binds a broad repertoire of ligands including
A.beta. (Stern et al., 2002). In mature animals there is relatively
little expression of RAGE in most tissues, whereas deposition of
ligands triggers RAGE expression. When A.beta. accumulates in AD or
in animal models of AD, RAGE expression increases particularly in
cerebral microvessels, a site of the BBB in vivo (Yan et al., 1996;
Deane et al., 2003; LaRue et al., 2004; Donahue et al., 2004). RAGE
binds soluble A.beta. in the nano-molar range, and mediates
pathophysiologic cellular responses consequent to ligation by
A.beta. (Yan et al., 1996; Mackic et al., 1998; Yan et al., 2000).
These include transport of pathophysiologically relevant
concentrations of plasma A.beta. across the BBB, neurovascular
stress, and reduction in the cerebral blood flow (CBF) (Deane et
al., 2003; LaRue et al., 2004). Deletion of the RAGE gene protects
the CNS pool of A.beta. from influences of its peripheral pool by
eliminating re-entry of circulating A.beta. into the brain, whereas
systemic treatment with soluble RAGE (sRAGE) sequesters circulating
A.beta. and reduces brain accumulation and deposition of A.beta. in
a mouse model of AD (Deane et al., 2003). Thus, compounds which
block A.beta./RAGE interaction at the BBB may also block re-entry
of A.beta. to the brain, reduce A.beta.-related pathology, and
improve CBF dysregulation and cognitive decline, which should have
important beneficial therapeutic effects in AD.
[0006] Compounds to inhibit A.beta./RAGE interaction, compositions
containing one or more of those compounds, and methods of treatment
are taught herein to be applicable to Alzheimer's disease and other
conditions involving A.beta.-RAGE interaction at the blood-brain
barrier, RAGE-mediated transport of A.beta. into the brain, and/or
RAGE activation RAGE activation in brain vasculature or brain
parenchyma. Other advantages of the invention are discussed below
or would be apparent to a person skilled in the art from that
discussion.
SUMMARY OF THE INVENTION
[0007] The invention is used to inhibit specific receptor-ligand
interaction between A.beta. and RAGE. This may be used to
manufacture a medicament (e.g., therapeutic and/or prophylactic
composition) to treat Alzheimer's disease or other conditions
involving A.beta.-RAGE interaction at the blood-brain barrier,
RAGE-mediated transport of A.beta. into the brain, and/or RAGE
activation RAGE activation in brain vasculature or brain
parenchyma. The amount or concentration A.beta. in the brain is
decreased, neuropathology associated with Alzheimer's disease is
reduced, cerebral blood flow is increased, memory and/or learning
is improved, or any combination thereof. In particular, compounds
such as tertiary amides R.sub.1(CO)NR.sub.2R.sub.3 and compositions
containing one or more compounds are considered embodiments of the
invention. R.sub.1 may be an electron-deficient aryl moiety (e.g.,
aryl moiety substituted with one or more electron withdrawing
groups, such as mono- or di-halide and mono- or di-nitro). R.sub.2
may be a hydrophobic hydrocarbon moiety (e.g., straight or branched
aliphatic, alicyclic such as cyclohexyl, straight or branched
acyclic or cyclic olefin, straight or branched acyclic or cyclic
acetylene, aryl-containing, and/or amine-containing C4-C18
hydrocarbon moiety which is optionally substituted). R.sub.3 may be
an electron-rich aryl moiety (e.g., furanyl, pyrrolyl, benzyl,
phenyl, alkylphenyl, alkoxy-phenyl, naphthyl, benzofuryl, indyl, or
quinolyl moiety which is optionally substituted) with or without a
C1-C4 alkyl (e.g., straight or branched) or heteroaromatic linker.
For example, one or more compounds may be manufactured as a
medicament or in a pharmaceutical composition.
[0008] Further aspects of the invention will be apparent to a
person skilled in the art from the following detailed description
and claims, and generalizations thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows the chemical structures of FPS1
(3-chloro-N-[2-[cyclohexyl[[1-[(4-fluorophenyl)methyl]-1H-pyrrol-2-yl]met-
hyl]amino]-2-oxoethyl]-N-(3-ethoxypropyl)-2,2-dimethyl-(9Cl)-propanamide);
FPS2
(3,5-dinitro-N-phenyl-N-[3-[(3,5,5-trimethyl-1-oxohexyl)amino]propyl-
]-(9Cl)-benzamide); and FPS3
(4-chloro-N-[2-(1H-indo-3-yl)-ethyl]-3-nitro-N-(2-pentyl-3-phenyl-allyl)--
benzamide).
[0010] FIG. 2 shows that the FPS1, FPS2, and FPS3 compounds block
A.beta.40/RAGE interaction. (A) .sup.125I-A.beta.40 specific
binding on RAGE-transfected CHO cells (square) or mock-transfected
CHO cells (triangle) at 4.degree. C. Compounds FPS1, FPS2, and FPS3
are high-affinity competitive inhibitors of A.beta.-RAGE binding on
RAGE-transfected CHO cells as indicated by the low nM values of
their inhibitory constants (Ki) (B) and the respective Ki/Kd ratios
(C) (Kd is the binding constant of A.beta. to RAGE from FIG. 2A).
Thiobarbituric acid reactive substance (TBARS) generation (D) and
NF-.kappa.B activation (E) at 1 .mu.M A.beta.40 were inhibited in
the presence of FPS2 at 50 nM or 500 nM. (F) FPS2 blocked
A.beta.40/soluble RAGE interaction in a cell-free system in vitro,
as determined by ELISA. The statistical significance of differences
is shown in FIGS. 2B-2D and 2F. Data are mean.+-.s.e.m., n=3 per
group.
[0011] FIG. 3 shows transport of .sup.125I-A.beta.40 at 1.5 nM
(corresponding to the plasma concentration in APPsw.sup.+/- mice)
across the BBB in vivo in 5-6 month old APPsw.sup.+/- mice (closed
bars) and age-matched controls (open bar) determined with the
arterial brain perfusion technique in the absence or presence of
RAGE-specific IgG (.alpha.-RAGE, 20 .mu.g/ml) or FPS2 (4.8
.mu.g/ml). The statistical significance of differences is shown.
Data are mean.+-.s.e.m., n=3-4 per group.
[0012] FIG. 4 shows that FPS2 treatment improves functional outcome
in APPsw.sup.+/- mice. (A) Percent increase in cerebral blood flow
(CBF) in response to brain activation in mice treated with vehicle
(open bar) or FPS2 (closed bar). (B-C) novel object location (B)
and novel object recognition (C) expressed as percent exploratory
preference in APPsw.sup.+/- mice treated with vehicle (open bar) or
FPS2 (closed bar). FPS2 (1 mg/kg per day) was intraperitoneally
administered for two months beginning at the age of 8 months old
mice. Values are means.+-.s.e.m, n=5-6 per group.
[0013] FIG. 5 shows that resting cerebral blood flow changes in
APPsw.sup.+/- mice after FPS2 administration. Cerebral blood flow
changes expressed as a percentage of baseline recorded over a 90
min period in mice administered FPS2 (filled triangle) or vehicle
(filled square). FPS2 (1 mg/kg) was administered intravenously
(arrow) as a bolus in 7-9 months old mice. Values are
means.+-.s.e.m, n=3 per group.
[0014] FIG. 6 shows that FPS2 clears A.beta. from brains in
APPsw.sup.+/- mice. A-B, A.beta.40 and A.beta.42 levels in
hippocampus (A) cortex (B) of APPsw.sup.+/- mice treated with
vehicle (filled square) or FPS2 (filled triangle). FPS2 (1 mg/kg
per day) was administered intraperitoneally to mice for two months
beginning at the age of 8 months. Values are means.+-.s.e.m, n=5-6
per group.
[0015] FIG. 7 shows that FPS2 reduces amyloid load clears in brains
of APPsw.sup.+/- mice. Amyloid load hippocampus and cortex of
APPsw.sup.+/- mice treated with vehicle (open bars) or FPS2 (closed
bars). FPS2 (1 mg/kg per day), was administered intraperitoneally
to mice for two months beginning at the age of 8 months. Values are
means.+-.s.e.m, n=5-6 per group.
[0016] FIG. 8 illustrates construction of a combinatorial library
designed to test structure-activity relationships based on FPS1,
FPS2, and FPS3.
[0017] FIG. 9 shows the chemical structures of N1A2B1
(N-benzyl-4-chloro-N-cyclohexyl-benzamide); N1A2B2
(N-benzyl-2,4-dichloro-N-cyclohexyl-benzamide); N2A1B1
(4-chloro-N-(3-methyl-butyl)-N-phenethyl-benzamide); N2A2B2
(N-benzyl-2,4-dichloro-N-(3-methyl-butyl)-benzamide); N2A2B3
(N-benzyl-N-(3-methyl-butyl)-3,5-dinitro-benzamide); N2A2B4
(N-benzyl-N-(3-methyl-butyl)-3-nitro-benzamide); N2A2B5
(N-benzyl-4-chloro-N-(3-methyl-butyl)-3-nitro-benzamide); N3A2B2
(N-benzyl-2,4-dichloro-N-(1-ethyl-propyl)-benzamide); N3A2B3
(N-benzyl-N-(1-ethyl-propyl)-3,5-dinitro-benzamide); N3A2B5
(N-benzyl-4-chloro-N-(1-ethyl-propyl)-3-nitro-benzamide); N4A2B2
(N-benzyl-2,4-dichloro-N-(3-phenyl-propyl)-benzamide); and N4A2B5
(N-benzyl-4-chloro-3-nitro-N-(3-phenyl-propyl)-benzamide). The mass
for each compound is shown next to the structure.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0018] RAGE is a multiligand receptor in the immunoglobulin
superfamily which binds a broad repertoire of ligands including
neurotoxic A.beta.. RAGE biology is largely dictated by the
expression or accumulation of its ligands. In mature animals there
is relatively little expression of RAGE in most tissues, whereas
deposition of ligand triggers receptor expression. When pathogenic
A.beta. accumulates in Alzheimer's disease (AD) and/or in
transgenic mouse models of AD, RAGE expression increases in the
brain, particularly in cerebral microvessels, a site of the
blood-brain barrier (BBB) in vivo, as we and others have reported.
We showed that overexpression of RAGE at the BBB increases influx
of circulating A.beta. into the brain, which is associated with
expression of proinflammatory cytokines, neurovascular stress, and
reduction in the cerebral blood flow (CBF). Deletion of the RAGE
gene suppresses A.beta. influx (i.e., transport) across the BBB
into the brain, reduces neurovascular stress, and improves CBF
dysregulation. Systemic treatment with soluble RAGE (sRAGE)
sequesters circulating A.beta. prevents its transport and re-entry
into the brain and reduces its brain accumulation and deposition in
the mouse model of AD. Thus, we hypothesized that a compound which
blocks A.beta./RAGE interaction at the BBB will inhibit re-entry
and should have beneficial therapeutic effects in Alzheimer's
disease.
[0019] By a primary screen on RAGE-transfected Chinese hamster
ovary (CHO) cells, three high-affinity inhibitors of A.beta./RAGE
interaction were identified out of seven hits from a diverse
library of 5000 small organic compounds. These three compounds
(FPS1, FPS2, and FPS3) share several structural characteristics
(FIG. 1). All three are tertiary amides substituted with a large,
hydrophobic hydrocarbon moiety and a monosubstituted aromatic
moiety. Linkage to the monosubstituted aromatic is either direct
(FPS2) or via an alkyl (FPS1) or heteroaromatic (FPS3) spacer. Two
of the compounds also feature a highly electron-poor substituted
benzene ring (3-nitro, 4-chloro for FPS1; 3,5-dinitro for
FPS2).
[0020] FIG. 2A shows saturable binding of .sup.125I-A.beta.40 to
RAGE in RAGE-transfected CHO cells at 4.degree. C.; the Kd (binding
constant) was 75.+-.8 nM as we reported (Mackic et al., 1998).
There was no binding on mock-transfected cells. All inhibit
A.beta.-RAGE binding competitively with the Ki (inhibitory
constant) in descending order of affinity FPS2>FPS3>FPS1
(FIG. 2B) close to the Kd (binding constant) of A.beta.40 binding
to RAGE in RAGE-transfected CHO cells. Their respective Ki/Kd
ratios were 0.45, 1.6, and 2.6, respectively, (FIG. 2C) where Ki is
the compound's inhibitory constant and Kd is the A.beta.-RAGE
binding constant. We focused on FPS2 as the highest affinity
blocker. In RAGE-transfected CHO cells, FPS2 inhibits
A.beta./RAGE-induced oxidant stress (FIG. 2D) and translocation of
nuclear factor kappa B (NF-.kappa.B) (FIG. 2E). It also inhibits
A.beta./RAGE interaction in a cell-free system in vitro (FIG.
2F).
[0021] An arterial vascular brain perfusion technique (LaRue et
al., 2004) was used to determine whether FPS2 blocks RAGE-mediated
transport of circulating .sup.125I-labeled A.beta.40 across the BBB
in 4-6 month old APPsw.sup.+/- mice. We and others have reported
that expression of RAGE in brain microvessels in these mice is
increased by about 5-fold compared to the littermate controls
(Deane et al., 2003; Donahue et al., 2004). FIG. 3 shows that
RAGE-specific IgG at high concentration of 20 .mu.g/ml of plasma
arterial inflow inhibits by >80% A.beta. transport across the
BBB, whereas FPS2 at 4.8 .mu.g/ml abolishes A.beta. transport
across the BBB resulting in 100% inhibition (FIG. 3). This data
suggests that FPS2, and possibly its related compounds, have
potential to block re-entry and influx of circulating A.beta.
across the BBB in vivo, which in an animal model of AD should
reduce A.beta. brain accumulation and amyloid pathology and improve
CBF dysregulation and behavior.
[0022] Compounds of the invention may be used as a medicament or to
formulate a composition with one or more of the utilities disclosed
herein. A "pharmaceutical" composition further contains a
physiologically-acceptable vehicle and is produced under aseptic
conditions. The composition may further comprise components useful
for delivering the compound to its site of action. Choice and
addition of such vehicles and carriers to the composition are
within the level of skill in the art. Compounds or compositions may
be administered in vitro to cells in culture, in vivo to cells in
the body, or ex vivo to cells outside of the subject that may later
be returned to the body of the same subject or another. The subject
is a human in need of treatment (e.g., a patient affected by
Alzheimer's disease and its progression, or at risk for its
development) or an animal model for Alzheimer's disease and its
pathogenesis.
[0023] The pharmaceutical composition may be administered as a
formulation adapted for delivery to systemic circulation,
blood-brain barrier, brain vasculature, or brain parenchyma.
Alternatively, the composition may be administered to culture
medium. In addition to the active compound, such compositions may
contain physiologically-acceptable vehicles, carriers, and other
ingredients known to facilitate administration and/or enhance
uptake (e.g., saline, dimethyl sulfoxide, lipid, polymer,
affinity-based cell specific-targeting systems). The composition
may be incorporated in a gel, sponge, or other permeable matrix
(e.g., formed as pellets or a disk) and placed in proximity to the
endothelium for sustained, local release. It may be administered in
a single dose or in multiple doses which are administered at
different times.
[0024] The pharmaceutical composition may be administered by any
known route. By way of example, the composition may be administered
by a mucosal, pulmonary, topical, or other localized or systemic
route (e.g., enteral and parenteral). The term "parenteral"
includes subcutaneous, intra-arterial, intradermal, intramuscular,
intrathecal, intravenous, and other injection or infusion
techniques, without limitation. In particular, an effective amount
of one or more compounds is present where A.beta. and RAGE
interact.
[0025] Suitable choices in amounts and timing of doses,
formulation, and routes of administration can be made with the
goals of achieving a favorable response in the subject with
Alzheimer disease or at risk thereof (i.e., efficacy), and avoiding
undue toxicity or other harm thereto (i.e., safety). Therefore,
"effective" refers to such choices that involve routine
manipulation of conditions to achieve a desired effect.
[0026] A bolus administered over a short time once a day is a
convenient dosing schedule. Alternatively, the effective daily dose
may be divided into multiple doses for purposes of administration,
for example, two to twelve doses per day. Dosage levels of active
ingredients in a pharmaceutical composition can also be varied so
as to achieve a transient or sustained concentration of the
compound in a subject, especially in and around vascular
endothelium of the brain or systemic circulation, and to result in
the desired therapeutic or prophylactic response. But it is also
within the skill of the art to start doses at levels lower than
required to achieve the desired therapeutic or prophylactic effect
and to gradually increase the dosage until that effect is
achieved.
[0027] The amount of compound administered is dependent upon
factors known to a person skilled in the art such as bioactivity
and bioavailability of the compound (e.g., half-life in the body,
stability, and metabolism); chemical properties of the compound
(e.g., molecular weight, hydrophobicity, and solubility); route and
scheduling of administration; and the like. For systemic
administration, passage of the compound or its metabolite through
the blood-brain barrier should not be critical. It should be
understood that the specific dose level to be achieved for any
particular subject may depend on a variety of factors, including
age, gender, health, medical history, weight, combination with one
or more other drugs, and severity of disease.
[0028] The term "treatment" of Alzheimer disease refers to, inter
alia, reducing or alleviating one or more symptoms in a subject,
preventing one or more symptoms from worsening or progressing,
promoting recovery or improving prognosis, and/or preventing
disease in a subject who is free therefrom as well as slowing or
reducing progression of existing disease. For a given subject,
improvement in a symptom, its worsening, regression, or progression
may be determined by objective or subjective measure. Efficacy of
treatment may be measured as an improvement in morbidity or
mortality. Preventative methods (e.g., preventing development of
disease or the incidence of relapse) are also considered
treatment.
[0029] The amount which is administered to a subject is preferably
an amount that does not induce toxic effects which outweigh the
advantages which result from its administration. Further objectives
are to reduce in number, diminish in severity, and/or otherwise
relieve suffering from the symptoms of the disease as compared to
recognized standards of care.
[0030] Production of compounds according to present regulations
will be regulated for good laboratory practices (GLP) and good
manufacturing practices (GMP) by governmental agencies (e.g., U.S.
Food and Drug Administration and European Medicines Agency). This
requires accurate and complete record-keeping, as well as
monitoring of QA/QC. Oversight of patient protocols by agencies and
institutional panels is also envisioned to ensure that informed
consent is obtained; safety, bioactivity, appropriate dosage, and
efficacy of products are studied in phases; results are
statistically significant; and ethical guidelines are followed.
Similar oversight of protocols using animal models, as well as the
use of toxic chemicals, and compliance with regulations is
required.
[0031] RAGE at the BBB is the major influx receptor for A.beta.
mediating its transport, retention and accumulation in the brain,
cytokine response, and suppression of the CBF (Deane et al., 2003;
LaRue et al., 2004). Our studies here indicate that RAGE at the BBB
is a major therapeutic target for inhibiting the pathogenic
consequences of A.beta.-vascular interactions, including
development of cerebral amyloidosis. These data strongly support
our proposal that treatment of APPsw.sup.+/- mice with FPS2 or the
related compounds FPS3 and FPS1 will (1) prevent development of
.beta.-amyloidosis, (2) improve dysregulated CBF, and (3) improve
behavior. The inhibitory effect of FPS2 on RAGE-mediated transport
of A.beta. across the BBB in vivo in APPsw.sup.+/- mice was
characterized. The therapeutic effects of FPS2 on A.beta.
pathology, CBF dysregulation, and behavior in APPsw.sup.+/- mice
was also evaluated. Other compounds of the invention may be
similarly characterized for their beneficial effect(s) on
Alzheimer's disease.
[0032] According to the amyloid hypothesis, A.beta. accumulation in
the brain is a chief event contributing to pathogenesis AD (Hardy
& Selkoe, 2002). Recent evidence indicates that A.beta. within
the intravascular space is linked to A.beta. deposited in the
brain. This suggests that transport of A.beta. between the
blood-to-brain and brain-to-blood paths across the BBB regulates
the level of A.beta. in the brain (Deane et al., 2003; Deane et
al., 2004; Tanzi et al., 2004; Zlokovic, 2005, Holtzman &
Zlokovic, 2006).
[0033] At the BBB, RAGE mediates transport of A.beta. into the CNS
which is associated with neurovascular stress, accumulation of
A.beta. in the brain, and development of A.beta.-related pathology
(Deane et al., 2003; LaRue et al., 2004; Donahue et al., 2004).
Increased expression of RAGE in cerebral microvessels at the BBB in
AD and in animal models of AD may result in increased transport of
plasma A.beta. into the brain, reduction in CBF, and expression of
proinflammatory cytokines. Deletion of the RAGE gene eliminates
influx of circulating A.beta. into the brain as well as
A.beta.-induced changes in CBF and inflammation. A soluble form of
RAGE (sRAGE) prevents accumulation of A.beta. in the brain in a
mouse model of AD. We hypothesize here that compounds which inhibit
RAGE/A.beta. interaction at the BBB will act as A.beta.-lowering
agents by preventing A.beta. effects on transport,
neuroinflammation, hypoperfusion, and accumulation in the
brain.
[0034] The therapeutic effect of compounds of the invention can be
determined in an animal model of AD. APPsw.sup.+/- mice can be
treated with one or more compounds as described for sRAGE (see
Deane et al., 2003). Mice can be sacrificed to determine A.beta.
load, A.beta.40 and A.beta.42 levels, and soluble/insoluble
fractions. The results confirmed our hypothesis that the compounds
reduce A.beta. accumulation and amyloid load substantially.
APPsw.sup.+/- mice have reduced CBF responses to whisker
stimulation (laedecola et al., 1999) and reduced resting CBF (Deane
et al., 2003), whereas blocking A.beta./RAGE interaction increases
CBF in AD mice (Deane et al., 2003). The CBF responses to whisker
stimulation can be measured in APPsw.sup.+/- mice to determine the
effects of treatment on brain activation-induced increases in CBF.
A battery of behavioral tests can be performed: Barnes maze for
spatial learning and memory (Bach et al., 1995); force-plate
actometer for general activity, spatial patterning, locomotion,
ataxia, tremor, and seizure (Zarcone, 2001); and operant chamber
for attention and spatial alternation in learning and memory
(Markowski et al., 2000).
[0035] Although toxicity of the compounds has not be observed in
assays (i.e., TUNEL, Hoechst staining, LDH release, negative
WST-8), it is possible that one or all compounds are toxic in vivo.
To determine if compounds are toxic in vivo, blood counts,
hemoglobin level, electrolytes, glucose, blood pressure, heart
rate, respiration, blood gasses, and pH will be determined. Kidney
function (urea, creatinine), liver enzymes, general activity,
spatial patterning, locomotion, ataxia, tremor, attention, and
spatial alternation in learning and memory may also be tested. It
is possible that compounds are not specific inhibitors of
A.beta.-RAGE binding. To address this issue, in vitro analysis may
be performed using a cell-free system with immobilized LRP,
apolipoproteins E and J, .alpha.2-macroglobulin, and other possible
receptors and binding proteins for A.beta. to confirm the
specificity of the compounds to prevent or disrupt A.beta.-RAGE
binding.
[0036] APPsw.sup.+/- mice were treated with FPS2 (1 mg/kg) or with
vehicle daily by intraperitoneal injection for two months starting
at 8 months old. At the end of the treatment period functional
changes in cerebral blood flow (CBF) to brain activation, and
memory tests were determined. Brain and plasma A.beta. levels were
also determined in FPS2 and vehicle treated mice. Compared to
vehicle chronic treatment with FPS2 increased CBF during brain
activation by about 50%, and significantly increased memory
determined by novel object location and novel object recognition
(FIG. 4). To establish whether FPS2 changes resting CBF, acute
studies were also conducted. In these studies, an intravenous bolus
injection of FPS2 (1 mg/kg) in APPsw.sup.+/- (7-9 months old)
transiently increased resting CBF by about 35% and peaked after
25-30 min (FIG. 5).
[0037] In the chronic-treated mice, A.beta.40 and A.beta.42 levels
in the hippocampus were reduced by about 85% and 90%, respectively,
in the FPS2-treated mice compared to vehicle (FIG. 6A). Similarly,
A.beta.40 and A.beta.42 levels in the cortex were reduced by about
70% and 80%, respectively (FIG. 6B). Thioflavin-S positive areas
(amyloid load) in the hippocampus and cortex were significantly
reduced by about 85% in the FPS2 treated mice compared to vehicle
(FIG. 7).
[0038] It is possible that the initial three compounds are not
effective enough in inhibiting A.beta./RAGE interaction and that
other compounds in the family will be more effective. Therefore,
building on the structural commonalities of FPS1 and FPS2, a
structure-activity study was carried out by synthesizing and
analyzing a library of 125 structurally-related compounds (see FIG.
8). For example, the tertiary amides R.sub.1(CO)NR.sub.2R.sub.3 may
be a product of R.sub.1 (e.g., 2-fluorobenzene, 4-fluorobenzene,
2,4-difluorobenzene, 3-chloro-4-fluorobenzene,
4-chloro-3-fluorobenzene, 3,5-difluorobenzene); R.sub.2 (e.g.,
ethyl, propyl, butyl, dimethyl-propane, hexyl, heptyl, octyl);
R.sub.3 (o-methoxy benzene, p-methoxy benzene,
2,4-dimethoxybenzene, 3,4-dimethoxybenzene,
3,4,5-trimethoxybenzene, 4-trifluoromethylbenzene); or any
combination thereof. These studies should result in discovery of
new A.beta. lowering agent(s) which can inhibit RAGE-A.beta.
interaction at the BBB and its pathophysiological consequences
including net blood-to-brain transport of A.beta.,
neuroinflammation, and accumulation of A.beta. in the brain.
[0039] Compounds were synthesized in parallel using semiautomated
chemical reactions. Five commercially-available aromatic aldehydes
were condensed in reductive amination reactions with five
hydrophobic amines to form 25 secondary amines. Parallel acylation
of these 25 secondary amines with five aromatic acyl chlorides
provided the final 125-compound library of tertiary amides. The
identity of compounds was verified by mass spectrometry (FIG. 9).
Purity was assessed by reverse-phase gradient HPLC. Library members
isolated with purity <95% were subjected to preparative HPLC
purification prior to assay.
[0040] Animals. APPsw.sup.+/- (Tg2576) mice were from Taconic Farms
(Germantown, N.Y.). Mice were housed under standard conditions (12
hour light/dark cycle starting at 07:00 AM; 21.+-.2.degree. C.;
55.+-.10% humidity) in solid-bottom cages on woodchip bedding. Mice
were anesthetized by intraperitoneal injection with urethane (750
mg/kg) and .alpha.-chloralose (50 mg/kg). All procedures on mice
were performed according to the NIH guidelines, which were approved
by the University of Rochester's Committee on Animal Resources.
[0041] FPS2 chronic treatment of APPsw.sup.+/- mice. APPsw.sup.+/-
mice (n=5-6 per group) were treated intraperitoneally with FPS2 (1
mg/kg per day) or saline for two months beginning at the age of 8
months. After two months of treatment, (i) the cerebral blood flow
response to brain activation (whisker stimulation) was measured and
(ii) behavioral tests including operant learning, novel object
location (NOL), and novel object recognition (NOR) were performed.
After these functional tests, the mice were sacrificed and their
brains analyzed for A.beta.40, A.beta.42, and amyloid levels.
[0042] CBF response to brain activation. The CBF response to
vibrissal stimulation in anesthetized APPsw.sup.+/- mice was
measured by using laser-doppler flowmetry (Transonic Systems, BLF
21 D). The tip of the laser-doppler probe was stereotaxically
placed 0.5 mm above the dura of the cranial window. The right
vibrissae were cut to about 5 mm and stimulated by gentle stroking
at 3-4 Hz, for 1 min with a cotton-tip applicator. Rectal
temperature was maintained at 37.degree. C. using a heated blanket
(Homeothermic Blanket, Harvard Apparatus). The percentage increase
in CBF due to vibrissal stimulation was obtained from the baseline
CBF and averaged for the three trials.
[0043] Memory testing. The memory tests consisted of three phases:
habituation, sample, and choice trials. Mice were first habituated
to the empty test box. A sample trial (object exposure) consisted
of placing a mouse into the test box which contained five different
objects. The mouse was removed from the test box and after a delay
(retention period) the mouse was placed back into the test box for
a choice trial. A choice trial could consist of switching the
location of two of the objects (Novel-Object Location (NOL) trial)
or substituted one object with a new object (Novel-Object
Recognition (NOR) trial). The time exploring the novel and familiar
objects was scored by hand to provide discrimination.
[0044] Human A.beta.40 and A.beta.42. APPsw.sup.+/- mice were
transcardially perfused with ice-cold heparinized saline and the
brains removed. Hippocampus and cortex were homogenized in ice-cold
guanidine buffer (5 M guanidine hydrochloride/50 mM TrisCl, pH 8.0)
and used for human A.beta.40 and A.beta.42 determination. Briefly,
brain (hippocampus and cortex) and plasma human A.beta.40 and
A.beta.42 levels were determined using human specific ELISA kits
KHB3481 and KHB3441, respectively, according to the manufacturer's
instructions (Invitrogen, Carlsbad, Calif.). For human A.beta.40
and A.beta.42 ELISA assays, a monoclonal antibody specific against
the NH.sub.2-terminus of human A.beta. was used as capturing
antibody and a rabbit antibody specific for the COOH-terminus of
either A.beta.40 or A.beta.42 was used as detecting antibody.
[0045] Amyloid load. Acetone-fixed cryostat brain slices (10 .mu.m)
were incubated with PBS containing 1% thioflavin S (Sigma Aldrich)
and subsequently washed with 80% ethanol, 90% ethanol, and
ddH.sub.2O. The thioflavin S-positive amyloid load was determined
using the IMAGE-PRO.RTM.-Plus software (Media Cybernetics).
[0046] FPS2 acute treatment of APPsw.sup.+/- mice. Resting CBF over
the sensory-motor cortex was determined using laser-doppler
flowmetry (Transonic Systems, BLF 21 D). The tip of the
laser-doppler probe was stereotaxically placed 0.5 mm above the
dura of the cranial window. 15 min after steady-state conditions
was obtained FPS2 (1 mg/kg) or vehicle was administered
intravenously (via the femoral vein) and CBF recorded for an
additional 75 min. Rectal temperature was maintained at 37.degree.
C. using a heated blanket (Homeothermic Blanket, Harvard
Apparatus). The percentage change in CBF from baseline was
obtained. Values were expressed as mean.+-.s.e.m. (n=3).
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[0080] Patents, patent applications, books, and other publications
cited herein are incorporated by reference in their entirety.
[0081] The term "about" may refer to the statistical uncertainty
associated with a measurement or the variability in a numerical
quantity which a person skilled in the art would understand does
not affect operation of the invention or its patentability.
[0082] All modifications and substitutions that come within the
meaning of the claims and the range of their legal equivalents are
to be embraced within their scope. A claim which recites
"comprising" allows inclusion of other elements to be within the
scope of the claim; the invention is also described by such claims
reciting the transitional phrases "consisting essentially of"
(i.e., allowing the inclusion of other elements to be within the
scope of the claim if they do not materially affect operation of
the invention) or "consisting of" (i.e., allowing only the elements
listed in the claim other than impurities or inconsequential
activities which are ordinarily associated with the invention)
instead of the "comprising" term. Any of these three transitions
can be used to claim the invention.
[0083] It should be understood that an element described in this
specification should not be construed as a limitation of the
claimed invention unless it is explicitly recited in the claims.
Thus, the granted claims are the basis for deter-mining the scope
of legal protection instead of a limitation from the specification
which is read into the claims. In contradistinction, the prior art
is explicitly excluded from the invention to the extent of specific
embodiments that would anticipate the claimed invention or destroy
novelty.
[0084] Moreover, no particular relationship between or among
limitations of a claim is intended unless such relationship is
explicitly recited in the claim (e.g., the arrangement of
components in a product claim or order of steps in a method claim
is not a limitation of the claim unless explicitly stated to be
so). All possible combinations and permutations of individual
elements disclosed herein are considered to be aspects of the
invention. Similarly, generalizations of the invention's
description are considered to be part of the invention.
[0085] From the foregoing, it would be apparent to a person of
skill in this art that the invention can be embodied in other
specific forms without departing from its spirit or essential
characteristics. The described embodiments should be considered
only as illustrative, not restrictive, because the scope of the
legal protection provided for the invention will be indicated by
the appended claims rather than by this specification.
* * * * *